1. Field of the Invention
The present invention has to do with a method of manufacturing D-tagatose from cheese whey and/or milk. More particularly, the invention relates to a method of producing D-tagatose by employing L-arabinose isomerase in the enzymatic isomerization of D-galactose recovered from the fermentation of a lactose hydrolysate. The lactose hydrolysate is derived from cheese whey and/or milk. D-tagatose is useful as a low calorie sweetener or bulking agent.
2. Description of Related Art
D-tagatose is the keto sugar of D-galactose. It has a sweetness equivalent to fructose but is poorly digested. Thus it can be used as a reduced calorie food sweetening and bulking agent. D-tagatose is also useful as an intermediate for the synthesis of other optically active compounds and as an additive in detergent, cosmetic and pharmaceutical formulations. D-tagatose is non-cariogenic and reduces insulin demand.
D-tagatose is presently made from D-galactose by chemical synthesis. The D-galactose is derived from lactose hydrolysate, which comprises galactose and glucose, two aldo-sugars which are difficult to separate. The galactose component is presently separated from the hydrolysate by column separation. The processes of chemical synthesis of D-tagatose and column separation of galactose are expensive, complicated and inefficient.
A current chemical process for synthesizing D-tagatose is described in U.S. Pat. No. 5,002,612. The process is carried out by isomerizing a mixture containing D-galactose with a metal hydroxide in the presence of an inorganic salt catalyst to form an intermediate metal hydroxide-D-tagatose complex. The intermediate then is neutralized with acid to yield D-tagatose.
The lack of suitable industrial methods for producing D-tagatose is recognized in the art as noted in Japanese patent disclosure 60-248196. The disclosure describes the production of D-tagatose from dulcitol by contacting a bacterial strain of the genus Arthrobacter with an aqueous solution containing dulcitol and recovering accumulated D-tagatose. This method has the disadvantage that dulcitol is not available in large quantities and is expensive. Moreover, the enzyme galactitol dehydrogenase requires NAD (nicotinimide-adenine dinucleotide) an expensive co-enzyme which makes the conversion of dulcitol to D-tagatose more costly.
Enzymatic methods for converting an aldose or aldose derivative to a ketose or ketose derivative are well known. The enzymatic conversion of glucose to fructose, for example, is widely practiced on a commercial scale. Enzymatic methods for converting D-galactose to D-tagatose, however, have not been developed until recently, as disclosed in the parent of this application.
The L-arabinose isomerase used according to the present invention is known for its use in the production of L-ribulose from L-arabinose. U.K. Patent Specification No. 1 497 888 (equivalent to U.S. Pat. No. 4,069,104) alleges that the conversion of D-galactose to D-tagatose using L-arabinose isomerase is known, but cites an unrelated reference (J. Biol Chem, 1071, 246, 5102-6) as a basis for the disclosure. No description of the conditions for such conversion are provided.
References having specific data concerning the activity of L-arabinose isomerase on D-galactose describe the activity as poor or undetectable. For example, D-galactose is reported as a poor substrate for propagating L-arabinose isomerase in a chapter entitled "L-Arabinose Isomerase" by K. Yamanaka and W. A. Wood, Methods In Enzymology, Vol. IX Carbohydrate Metabolism at 596-602 (Edited by Willis A. Wood, 1966). L-arabinose isomerase derived from Lactobacillus gayonii was found to have a high affinity for L-arabinose, but low affinity for D-galactose and D-fucose. The authors concluded that the isomerase had affinity for sugars with an L-cis hydroxyl configuration at C-3 and C-4. Id. at 602.
The activity of L-arabinose isomerase on D-galactose is further reported as "slight" in "Crystallization and Properties of L-Arabinose Isomerase From Lactobacillus Gayonii", Nakamatu, Biochim. Biophys. Acta, 178 (169) at 156-165. It is also described as a "slow" substrate for L-arabinose-L-ribulose-isomerase in "Degradation of L-Arabinose by Aerobacter Aerogenes", F. J. Simpson, The Journal of Biological Chemistry, 1958, Volume 230, pages 457-472.
A comparison of L-arabinose isomerases from Mycobacterium smegmatis induced by L-arabinose and D-galactose is reported by K. Izumori et al. in Journal of Bacteriology, January 1978 at 413-414. D-galactose was found to induce both an L-arabinose permease and an L-arabinose isomerase. The isomerase induced by D-galactose had similar properties to the L-arabinose induced isomerase. D-galactose was found to be suitable as a substrate of the permease, but not a substrate of the isomerase. Id. at 414.
Purified galactose is difficult to make because it must be separated from glucose, both aldo-sugars. Mixtures of D-galactose and D-glucose are produced when lactose is hydrolyzed by lactase. The lactose can be derived from cheese whey and/or milk. A method of preparing galactose from lactose is disclosed in U.S. Pat. No. 4,595,659 to Roland et al.
An excess of lactose is currently produced by the North American dairy industry as a by product of cheese manufacture, in the form of whey, whey permeate or milk permeate. These by-products are a potential source of food for both human and animal consumption and methods for using them are being sought. The enzymatic hydrolysis of lactose to glucose and galactose by beta-galactosidase (also referred to in this specification as lactase, beta-galactosidase being the systemic name for lactase), followed by fermentation, is described by J. R. Ernandes et al., "Simultaneous Utilization of Galactose and Glucose by Saccharomyces spp.", Biotechnology Technique, Vol. 6, No. 3, May/June 1992, pp. 233-238. The authors describe a means of adapting yeast for galactose utilization to allow the simultaneous uptake of galactose and glucose, thereby overcoming the tendency of glucose to be utilized by yeast cells in preference to other sugars.
Other processes for using whey or lactose as fermentation substrates have been described in the literature. A process for co-fermenting ethanol from cheese whey and corn using fungal alpha-amylase is described by P. J. Whaler, "Development, Scale-up and a Continuous Fermentation Process for the Cofermentation of Cheese Whey and Corn for Ethanol Production," University of Nebraska, Lincoln, Nebr. 68583-0745 U.S.A., Dissertation Abstracts International, B1988, 49(4) 964-965.
The production of a fermented beverage using lactase hydrolyzed milk was reported by T. Miyamoto et al., Japanese Journal of Dairy and Food Science, 1986, 35(4) A143-A150. Whey and skim milk were incubated with beta-galactosidase from Aspergillus oryzae. The process gave 80-90% hydrolysis of lactose in whey and 40-50% hydrolysis in skim milk. Production of acid and ethanol by Zymomonas mobilis, ATCC 10,988, incubated in whey for 36 hours at 30.degree. C. was very slight, but increased as the degree of lactose hydrolysis increased. Chromatographic analysis showed that glucose in the treated whey was almost completely utilized by Z. mobilis within 18 hours, but that galactose and lactose were hardly affected. Mixed cultures of Z. mobilis with various Streptococcus and Lactobacillus enzymes were found to increase acid production but did not increase the amount of ethanol produced by Z. mobilis, the maximum being 0.88% in lactose-hydrolyzed whey.
In a comparison of lactose-hydrolyzed whey and normal whey as substrates for alcohol production, galactose was found to be a poor substrate for alcohol production. V. S. O'Leary et al., "Influence of Lactose Hydrolysis and Solids Concentration on Alcohol Production by Yeast in Acid Whey Ultrafiltrate," Biotechnology and Bioengineering, Vol. XIX, pages 1689-1702 (1977). Saccharomyces cerevisiae and Kluyveromyces fragilis were tested. S. cerevisiae was found to give higher alcohol yields, but the process was deemed wasteful because galactose, which comprised about half the available carbohydrate, was not fermented.
The fermentation of glucose has been reported as a means of enriching fructose. A method of producing very enriched fructose syrup by selective conversion of glucose to ethanol using immobilized cells of S. cerevisiae, ATCC 36,859 (mutated culture), is described by D. W. Koren et al., "Continuous Production of Fructose Syrup and Ethanol from Hydrolysed Jerusalem Artichoke Juice," Journal of Industrial Microbiology, 7 (1991) pages 131-136. Some fructose was consumed and the glucose/fructose conversion rate ratio was regulated by the glucose concentration in the product stream. The same S. cerevisiae was used to produce sorbitol and ethanol from Jerusalem artichokes. Z. Duvnjak et al., "Production of Sorbitol and Ethanol from Jerusalem Artichokes by Saccharomyces cerevisiae ATCC 36859," Applied Microbiology Biotechnology (1991) 35:711-715. The sorbitol was produced from fructose following ethanol production from glucose.
None of the foregoing literature references or patents disclose or suggest an enzymatic process for isomerizing D-galactose to D-tagatose. Moreover, one skilled in the art would be taught away from using L-arabinose isomerase for the isomerization because the art describes the activity of the enzyme on D-galactose as poor.